Method of treating hyperproliferative diseases using active vitamin D analogues

ABSTRACT

Methods for the utilization of hypocalcemic vitamin D analogs to inhibit the hyperproliferation of malignant or neoplastic cells without incidence of hypercalcemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/596,149, filed Feb. 23, 1998, which is a divisional of U.S.application Ser. No. 08/781,910, filed Dec. 30, 1996, now U.S. Pat. No.5,763,429 all of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

BACKGROUND OF THE INVENTION

This invention relates generally to a method of treatinghyperproliferative diseases, and in particular, to the use of activeforms of hypocalcemic vitamin D to inhibit the hyperproliferativecellular activity of these diseases and to promote differentiation ofthe cells.

Extensive research during the past two decades has established importantbiologic roles for vitamin D apart from its classic role in bone andmineral metabolism. Specific nuclear receptors for1α,25-dihydroxyvitamin D₃, the hormonally active form of vitamin D, arepresent in cells from diverse organs not involved in calciumhomeostasis. For example, specific, biologically active vitamin Dreceptors have been demonstrated in the human prostatic carcinoma cellline, LNCaP, (Miller et al., 52 Cancer Res. (1992) 515-520); Vitamin Dreceptors have also been described for many other neoplastic cells,e.g., carcinomas of the breast and the colon.

It has been reported that certain vitamin D compounds and analogues arepotent inhibitors of malignant cell proliferation and areinducers/stimulators of cell differentiation. For example, U.S. Pat. No.4,391,802 issued to Suda et al. discloses that 1α-hydroxyvitamin Dcompounds, specifically 1α,25-dihydroxyvitamin D₃ and 1α-hydroxyvitaminD₃, possess potent antileukemic activity by virtue of inducing thedifferentiation of malignant cells (specifically leukemia cells) tononmalignant macrophages (monocytes), and are useful in the treatment ofleukemia. Antiproliferative and differentiating actions of1α,25-dihydroxyvitamin D₃ and other vitamin D₃ analogues have beenreported with respect to cancer cell lines. More recently, anassociation between vitamin D receptor gene polymorphism and cancer riskhas been reported, suggesting that vitamin D receptors may have a rolein the development, and possible treatment, of cancer.

These previous studies have focused exclusively on vitamin D₃ compounds.Even though these compounds may indeed be highly effective in promotingdifferentiation in malignant cells in culture, their practical use indifferentiation therapy as anticancer agents is severely limited becauseof their equally high potency as agents affecting calcium metabolism. Atthe levels required in vivo for effective use as, for example,antileukemic agents, these same compounds can induce markedly elevatedand potentially dangerous blood calcium levels by virtue of theirinherent calcemic activity. That is, the clinical use of1α,25-dihydroxyvitamin D₃ and other vitamin D₃ analogues as anticanceragents is precluded, or severely limited, by the risk of hypercalcemia.This indicates a need for compounds with greater specific activity andselectivity of action, i.e., vitamin D compounds with antiproliferativeand differentiating effects but which have less calcemic activity.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method of treating hyperproliferativedisease conditions such as those characterized by hyperproliferativecell growth and/or abnormal cell differentiation. The method includesuse of active vitamin D compounds to inhibit abnormal cell growth andpromote cell differentiation.

The foregoing, and other advantages of the present invention, arerealized in one aspect thereof in a method of inhibiting thehyperproliferative activity of neoplastic or hyperplastic cells,comprising treating the cells with an effective amount of a hypocalcemicvitamin D compound. The treating step includes inhibiting proliferationof, and inducing and enhancing differentiation in such cells.

The hypocalcemic vitamin D compounds of the present invention includevitamin D compounds having a hydrocarbon moiety substituted at the C-24position on the sidechain of the molecule and a hydroxy groupsubstituted in at least one of the C₁, C₂₄ or C₂₅ positions.

The vitamin D compound of the present invention is an active vitamin Dand is suitably represented by the formula (I) described hereafter. Thecompounds of formula (I) suitably include 1α,24-dihydroxyvitamin D₂,1α,24-dihydroxyvitamin D₄, 1α,25-dihydroxyvitamin D₄,1α,25-dihydroxyvitamin D₂, 1α-hydroxyvitamin D₂ and 1α-hydroxyvitaminD₄.

Hypocalcemic vitamin D compounds are valuable for the treatment ofbreast and colon cancer, as well as other neoplasms such as pancreaticcancer, endometrial cancer, small cell and non-small cell cancer of thelung (including squamous, adneocarcinoma and large cell types), squamouscell cancer of the head and neck, bladder, ovarian and cervical cancers,myeloid and lymphocyltic leukemia, lymphoma, hepatic tumors, medullarythyroid carcinoma, multiple myeloma, melanoma, retinoblastoma, andsarcomas of the soft tissue and bone.

In accordance with the present invention, when effective amounts ofhypocalcemic vitamin D compounds are administered to patients withcancer or neoplasms, the proliferative activity of the abnormalneoplastic cells is inhibited, reduced, or stabilized, and celldifferentiation is induced, promoted or enhanced, with significantlyless hypercalcemia and hypercalciuria than is observed after the sameamount of an activated vitamin D₃ (e.g., 1α-OH D₃, 1α,25-(OH)₂ D₃) isadministered in previously known formulations. Thus, the compound inaccordance with the present invention has an improved therapeutic indexrelative to active forms of vitamin D₃ analogues.

Accordingly, another aspect of the invention is a method of treatinghuman cancer comprising administering to a subject who has cancer aneffective amount of hypocalcemic vitamin D compound which has or attainsthrough metabolism in vivo, a vitamin D receptor (VDR) binding affinitysubstantially equivalent to the binding affinity of1α,25-dihydroxyvitamin D₃ and a hypercalcemia risk substantially lowerthat that of 1α,25-dihydroxyvitamin D₃, to inhibit, decrease orstabilize the cellular abnormal proliferative activity of the cancer.

For treatment for malignant conditions in accordance with the presentinvention, the hypocalcemic vitamin D compounds can be suitablyadministered alone as an active ingredient, as an antiproliferativeagent in a pharmaceutical composition, or co-administered with ananticancer agent.

Further, included within the scope of the present invention is theco-administration of the vitamin D of formula (I) with a cytotoxic oranticancer agent. Such agents suitably include antimetabolites (e.g.,5-fluoro-uracil, methotrexate, fludarabine), antimicrotubule agents(e.g., vincristine, vinblastine, taxanes such as paclitaxel, docetaxel),an alkylating agent (e.g., cyclophasphamide, melphalan,biochoroethylnitrosurea, hydroxyurea), platinum agents (e.g. cisplatin,carboplatin, oxaliplatin, JM-216, CI-973), anthracyclines (e.g.,doxrubicin, daunorubicin), antibiolitics (e.g., mitomycin, idarubicin,adriamycin, daunomycin), topoisomerase inhibitiors (e.g., etoposide,camptothecins) or any other antineoplastic agents. (estramustinephosphate, prednimustine).

It is anticipated that the hypocalcemic vitamin D compounds used incombination with various anticancer drugs can give rise to asignificantly enhanced cytotoxic effect on cancerous cells, thusproviding an increased therapeutic effect. Specifically, as asignificantly increased growth-inhibitory effect is obtained with theabove disclosed combinations utilizing lower concentrations of theanticancer drugs compared to the treatment regimes in which the drugsare used alone, there is the potential to provide therapy whereinadverse side effects associated with the anticancer drugs areconsiderably reduced than normally observed with the anticancer drugsused alone in larger doses. Possible dose ranges of theseco-administered anticancer agents are about 0.1 to 20 mg/kg/day.

Also included within the scope of the present invention is theco-administration of effective dosages of the analogue of formula (I) inconjunction with administration of hormones or other agents, e.g.,estrogens, which are known to ameliorate bone diseases or disorders. Forexample, prostate cancer often metastasizes to bone, causing bone lossand associated pain. Such bone agents may include conjugated estrogensor their equivalents, calcitonin, bisphosphonates, calcium supplements,cobalamin, pertussis toxin and boron.

In another aspect, the invention is a pharmaceutical composition whichincludes an anticancer agent which is an active vitamin D compound; anagent selected from the group consisting of (i) an anticancer agent,(ii) a bone agent, and combinations thereof; and a physiologicallyacceptable carrier.

Other advantages and a fuller appreciation of specific adaptations,compositional variations, and physical attributes will be gained upon anexamination of the following detailed description of preferredembodiments, taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWING(S)

Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an effective method for the treatment ofneoplasms and hyperproliferative diseases. Particularly, the presentinvention relates to therapeutic methods for inhibiting, reducing orstabilizing the hyperproliferative cellular activity of diseased cells,and inducing, enhancing or promoting cell differentiation in thediseased cells. The present invention provides a novel treatment of apatient suffering from a hyperproliferative disease such as prostaticcancer or prostatic hyperplasia with a hypocalcemic hydroxyvitamin Danalogue. The vitamin D analogue is suitably a 1α-hydroxyvitamin D or a24-hydroxyvitamin D compound. The hypocalcemic hydroxyvitamin D analoguerepresented by formula (I) as described hereinbelow is provided to thepatient without causing dose-limiting hypercalcemia and hypercalciuria,i.e., unphysiologically high and deleterious blood calcium levels andurine calcium levels, respectively. These attributes are achievedthrough specific chemical properties of the hypocalcemic vitamin Dcompounds as described.

In accordance with the present invention, when effective amounts of thehypocalcemic vitamin D compounds are administered to patients withcancer or hyperplasia, the proliferative activity of the abnormal cellsis inhibited, maintained, or alleviated, and cell differentiation isinduced, promoted or enhanced, with significantly less hypercalcemia andhypercalciuria than is observed after the same amount of activatedvitamin D₃ is administered in previously known formulations. Thus, thehypocalcemic vitamin D compounds of the present invention have animproved therapeutic index relative to active forms of vitamin D₃analogues.

It is known that vitamin D₃ must be hydroxylated in the C-1 and C-25positions before it is activated, i.e., before it will produce abiological response. A similar metabolism appears to be required toactivate other forms of vitamin D, e.g., vitamin D₂ and vitamin D₄.Therefore, as used herein, the term “activated vitamin D” or “activevitamin D” is intended to refer to a vitamin D compound or analogue thathas been hydroxylated in at least the C-1, C-24 or C-25 position of themolecule and either the compound itself or its metabolites in the caseof a prodrug, such as 1α-hydroxyvitamin D₂, binds the vitamin D receptor(VDR). For example, vitamin D “prodrugs” include compounds which arehydroxylated in the C-1 position. Such compounds undergo furtherhydroxylation in vivo and their metabolites bind the VDR.

The term “hypocalcemic vitamin D compound” is in reference to activevitamin D analogs which demonstrate reduced calcemic activity relativeto the calcemic activity of 1α,25-dihydroxyvitamin D₃. Such compoundsinclude 24-hydroxyvitamin D compounds, 25-hydroxyvitamin D compounds and1α-hydroxyvitamin D compounds. The calcemic activity of these compoundsranges from 0.001 to 0.5 that of 1α,25-dihydroxyvitamin D₃.

Also, as used herein, the term “lower” as a modifier for alkyl, alkenylacyl, or cycloalkyl is meant to refer to a straight or branched,saturated or unsaturated hydrocarbon radical having 1 to 4 carbon atoms.Specific examples of such hydrocarbon radicals are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, t-butyl, ethenyl, propenyl, butenyl,isobutenyl, isopropenyl, formyl, acetyl, propionyl, butyryl orcyclopropyl. The term “aromatic acyl” is meant to refer to aunsubstituted or substituted benzoyl group.

As used herein, the term “hydrocarbon moiety” refers to a lower alkyl, alower alkenyl, a lower acyl group or a lower cycloalkyl, i.e., astraight or branched, saturated or unsaturated C₁-C₄ hydrocarbon radial.

The compound in accordance with the present invention is an activehypocalcemic vitamin D compound. Further, the active vitamin D inaccordance with the present invention may have an unsaturated sidechain,e.g., there is suitably a double bond between C-22 and C-23, betweenC-25 and C-26 or between C-26 and C-27.

A hypocalcemic hydroxyvitamin D of the present invention has the generalformula described in formula (I)

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso thatboth R¹ and R² cannot both be an alkenyl, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, X²is hydrogen or hydroxyl, or,may be taken with R¹ or R², to constitute a double bond, X³ is hydrogenor hydroxyl provided that at least one of X¹, X² and X³ is hydroxyl; andY is a methylene group if the bond to Y is a double bond or is a methylgroup or hydrogen if the bond to Y is a single bond.

A 1α-hydroxyvitamin D compound of formula (I) is characterized by thegeneral formula (II):

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso thatboth R¹ and R² cannot both be an alkenyl, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, X² is hydrogen or hydroxyl, or,may be taken with R¹ or R², to constitute a double bond, and Y is amethylene group if the bond to Y is a double bond or is a methyl groupor hydrogen if the bond to Y is a single bond.

Specific 1α-hydroxyvitamin D compounds in accordance with the presentinvention are characterized by the general formula (III):

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso thatboth R¹ and R² cannot both be an alkenyl, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, and X² is hydrogen or hydroxyl,or, may be taken with R¹ or R², to constitute a double bond.

The hypocalcemic vitamin D compounds of the present invention are thosethat have effective antiproliferative and cell differentiation activity(i.e., reversal of malignant transformation), but have a lower tendencyor inability to cause the undesired side effects of hypercalcemia and/orhypercalciuria. In other words, the compounds of the present inventioncan be administered at dosages that allow them to act asantiproliferative agents and cell differentiation agents when exposed tomalignant or other hyperproliferative cells without significantlyaltering calcium metabolism. This selectivity and specificity of actionmakes the hypocalcemic vitamin D compounds useful and preferred agentsfor safely inhibiting hyperproliferation and promoting malignant orhyperplastic cell differentiation. The compounds of the presentinvention, thus, overcome the shortcomings of the known active vitaminD₃ compounds described above, and can be considered preferred agents forthe control and treatment of malignant diseases such breast, prostate,testicular and colon cancer, as well as other neoplasms such aspancreatic cancer, endometrial cancer, small cell and non-small cellcancer of the lung (including squamous, adneocarcinoma and large celltypes), squamous cell of the head and neck, bladder, ovarian andcervical cancers, myeloid and lymphocyltic leukemia, lymphoma, hepatictumors, medullary thyroid carcinoma, multiple myeloma, melanoma,retinoblastoma, and sarcomas of the soft tissue and bone, i.e. neoplasmsthat express a vitamin D receptor.

Thus, the present invention provides a method of treating malignantcells as well as other hyperproliferative cells, (i.e., inhibiting theirhyperproliferative activity and/or inducing and enhancing theirdifferentiation) with an effective amount of a hypocalcemic vitamin Dcompound. The effective dosage amount on a daily basis per kilogram ofbody weight of the patient ranges from about 0.01 μg/kg/day to about 2.0μg/kg/day. The compounds in accordance with the present invention can begiven in daily dose or episodic dose, e.g., once every 2-6 days or oncea week, the dose in each day can be a single dose or divided into 2-4subdoses which can be given, e.g., an hour apart until the total dose isgiven. The compounds in accordance with the present invention areadministered in an amount that raises a serum vitamin D level to asupraphysiological level for a sufficient period of time to inducedifferentiation or regression of a tumor or neoplasm with causinghypercalcemia. The hypocalcemic properties of the compound permit suchsupraphysiologic levels.

The compounds of formula (I) are valuable for the treatment of cancerand neoplasms in a patient suffering therefrom. In particular, theinvention is a method for treating a patient suffering from thehyperproliferative cellular effects of cancer and other neoplasms byadministering to the patient a therapeutically effective amount of acompound of formula (I), which is suitably 1α,24-dihydroxyvitamin D₂,1α,24-dihydroxyvitamin D₄, 1α,25-dihydroxyvitamin D₂,1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitamin D₂, and 1α-hydroxyvitaminD₄. Among those compounds of formula (I) that have a chiral center inthe sidechain, such as at C-24, it is understood that both epimers(e.g., R and S) and the racemic mixture are within the scope of thepresent invention.

The compounds of formula (I) can be prepared as described, e.g., in U.S.Pat. No. 5,488,120 issued to Knutson et al., U.S. Pat. Nos. 4,554,106,4,670,190 and 5,486,636 issued to DeLuca et al., and Strugnell et al.,310 Biochem. J. (1995) pp. 233-241, all of which are incorporated hereinby reference.

The biopotencies of the compounds of formula(I) have been studied andcompared to that of 1α,25-dihydroxyvitamin D₃, the active hormonal formof vitamin D and the standard against which all vitamin D compounds andanalogues are measured. For example, it has been found that the vitaminD receptor (VDR) binding affinities of the compounds of formula (I), ortheir active metabolites, are substantially equivalent to (i.e., equalto or up to 3 times weaker than) the affinity of 1α,25-dihydroxyvitaminD₃. Such receptor binding affinities are indicative of potent biologicalactivity.

At the same time, it has been found that compounds of formula (I) aresignificantly less toxic than their corresponding vitamin D₃ analogues.For example, in parent co-pending application, Ser. No. 08/265,438, thedisclosure of which is incorporated herein by reference, the LD₅₀ for1α-hydroxyvitamin D₄ was found to be 1.0 mg/kg in males and 3.0 mg/kg infemales, i.e., substantially less toxic than 1α-hydroxyvitamin D₃(LD₅₀˜0.2 mg/kg). Further, in the parent U.S. Pat. No. 5,403,831, andits grandparent U.S. Pat. No. 5,104,864, both of which are incorporatedherein by reference, it has been shown that 1α-hydroxyvitamin D₂ has thesame biopotency as 1α-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃but is much less toxic. Even dosages up to 10 μg/day of1α-hydroxyvitamin D₂ in women with postmenopausal osteoporosis elicitedonly mild hypercalciuria (U.Ca>300 mg/24 hrs), and no markedhypercalcemia (S. Ca>11.0 mg/dL) solely due to 1α-hydroxyvitamin D₂ wasevident. Additionally, the compound did not adversely affect kidneyfunction, as determined by creatinine clearance and BUN; nor did itincrease urinary excretion of hydroxyproline, indicating the absence ofany stimulatory effect on bone resorption. Administration of1α-hydroxyvitamin D₂ to healthy adult males in dosages up to 8 μg/dayshowed no clinically significant hypercalcemia or other adverse effects.

The compounds of formula (I) are useful as active ingredients inpharmaceutical compositions having reduced side effects and low toxicityas compared with the known analogues of active forms of vitamin D₃.

The pharmacologically active compounds of this invention can beprocessed in accordance with conventional methods of pharmacy to producemedicinal agents for administration to patients, e.g., mammals includinghumans. For example, the hypercalcemic vitamin D compounds of thepresent invention can be employed in admixtures with conventionalexcipients, e.g., pharmaceutically acceptable carrier substancessuitable for enteral (e.g., oral), parenteral or topical applicationwhich do not deleteriously react with the active compounds.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions, alcohols, gum arabic, vegetable oils(e.g., almond oil, corn oil, cottonseed oil, peanut oil, olive oil,coconut oil), mineral oil, fish liver oils, oily esters such asPolysorbate 80, polyethylene glycols, gelatine, carbohydrates (e.g.,lactose, amylose or starch), magnesium stearate, talc, silicic acid,viscous paraffin, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc.

The pharmaceutical preparations can be sterilized and, if desired, bemixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, flavoring and/or one or more other activecompounds, for example, vitamin D₃ and its 1α-hydroxylated metabolites,conjugated estrogens or their equivalents, anti-estrogens, calcitonin,biphosphonates, calcium supplements, cobalamin, pertussis toxin andboron.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solution, as well assuspensions, emulsions, or implants, including suppositories. Parenteraladministration suitably includes subcutaneous, intramuscular, orintravenous injection, nasopharyngeal or mucosal absorption, ortransdermal absorption. Where indicated, the compounds of formula (I)may be given by direct injection into the tumor, e.g., parathyroidadenoma, or by regional delivery, e.g.,by intraarterial delivery ordelivery via the portal vein. Regional delivery is especially suitablefor treatment of hepatic cancers. Ampoules are convenient unit dosages.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, lozenges, powders, or capsules. A syrup,elixir, or the like can be used if a sweetened vehicle is desired.

For topical application, suitable nonsprayable viscous, semi-solid orsolid forms can be employed which include a carrier compatible withtopical application and having a dynamic viscosity preferably greaterthan water, for example, mineral oil, almond oil, self-emulsifyingbeeswax, vegetable oil, white soft paraffin, and propylene glycol.Suitable formulations include, but are not limited to, creams,ointments, lotions, solutions, suspensions, emulsions, powders,liniments, salves, aerosols, transdermal patches, etc., which are, ifdesired, sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, demulsifiers, wetting agents, etc. A cream preparation inaccordance with the present invention suitably includes, for example,mixture of water, almond oil, mineral oil and self-emulsifying beeswax;an ointment preparation suitably includes, for example, almond oil andwhite soft paraffin; and a lotion preparation suitably includes, forexample, dry propylene glycol.

Topical preparations of the compound in accordance with the presentinvention useful for the treatment of skin disorders may also includeepithelialization-inducing agents such as retinoids (e.g., vitamin A),chromanols such as vitamin E, β-agonists such as isoproterenol or cyclicadenosine monophosphate (cAMP), anti-inflammatory agents such ascorticosteroids (e.g., hydrocortisone or its acetate, or dexamethasone)and keratoplastic agents such as coal tar or anthralin. Effectiveamounts of such agents are, for example, vitamin A about 0.003 to about0.3% by weight of the composition; vitamin E about 0.1 to about 10%;isoproterenol about 0.1 to about 2%; cAMP about 0.1 to about 1%;hydrocortisone about 0.25 to about 5%; coal tar about 0.1 to about 20%;and anthralin about 0.05 to about 2%.

For rectal administration, the compound is formed into a pharmaceuticalcomposition containing a suppository base such as cacao oil or othertriglycerides. To prolong storage life, the composition advantageouslyincludes an antioxidant such as ascorbic acid, butylated hydroxyanisoleor hydroquinone.

For treatment of calcium metabolic disorders, oral administration of thepharmaceutical compositions of the present invention is preferred.Generally, the compound of this invention is dispensed by unit dosageform comprising about 0.5 μg to about 25 μg in a pharmaceuticallyacceptable carrier per unit dosage. The dosage of the compound accordingto this invention generally is about 0.01 to about 1.0 μg/kg/day,preferably about 0.04 to about 0.3 g/kg/day. Oral dosing for thetreatment of cancers and neoplasms and other hyperproliferative diseasesgenerally is about 10 μg to 200 μg/day.

For topical treatment of skin disorders, the dosage of the compound ofthe present invention in a topical composition generally is about 0.01μg to about 50 μg per gram of composition. For treatment of cancers, thedosage of the hypocalcemic vitamin D compound in a locally appliedcomposition generally is about 0.01 μg to 100 μg per gram composition.

Oral administration of the pharmaceutical compositions of the presentinvention is preferred. The dosage of the compounds for the treatment ofcancer or neoplasms according to this invention generally is about 0.01to about 2.0 μg/kg/day, preferably about 0.01 to about 1.0 μg/kg/day. Asnoted above, dosing of the hypocalcemic vitamin D compounds inaccordance with the present invention can be done on an episodic basis,in which higher does can be used, generally about 20 μg to about 200 μggiven once every 2-7 days. Generally, the compounds of this inventionare dispensed by unit dosage form in a pharmaceutically acceptablecarrier.

Those of ordinary skill in the art will readily optimize effective dosesand coadministration regimens as determined by good medical practice andthe clinical condition of the individual patient. Regardless of themanner of administration, it will be appreciated that the actualpreferred amounts of active compound in a specific case will varyaccording to the efficacy of the specific compound employed, theparticular compositions formulated, the mode of application, and theparticular situs and organism being treated. For example, the specificdose for a particular patient depends on age, body weight, general stateof health, on diet, on the timing and mode of administration, on therate of excretion, and on medicaments used in combination and theseverity of the particular disorder to which the therapy is applied.Dosages for a given host can be determined using conventionalconsiderations, e.g., by customary comparison of the differentialactivities of the subject compounds and of a known agent, such as bymeans of an appropriate conventional pharmacological protocol.

Further, included within the scope of the present invention is a methodof co-administration of hypercalemic vitamin D compounds with ananticancer or antineoplastic agent. Such agents may suitably includeantimetabolites (e.g., 5-fluorouracil, methotrexate, fludarabine),antimicrotubule agents (e.g., vincristine, vinblastine, taxanes such aspaclitaxel, docetaxel), an alkylating agent (e.g., cyclophasphamide,melphalan, biochoroethylnitrosurea, hydroxyurea), platinum agents (e.g.cisplatin, carboplatin, oxaliplatin, JM-216, CI-973), anthracyclines(e.g., doxrubicin, daunorubicin), antibiolitics (e.g., mitomycin,idarubicin, adriamycin, daunomycin), topoisomerase inhibitiors (e.g.,etoposide, camptothecins) or any other antineoplastic agents.(estramustine phosphate, prednimustine). It is anticipated thathypercalcemic vitamin D compounds used in combination with variousanticancer drugs can give rise to a significantly enhanced cytotoxiceffect on cancerous cells, thus providing an increased therapeuticeffect. Specifically, as a significantly increased growth-inhibitoryeffect is obtained with the above disclosed combinations utilizing lowerconcentrations of the anticancer drugs compared to the treatment regimesin which the drugs are used alone, there is the potential to providetherapy wherein adverse side effects associated with the anticancerdrugs are considerably reduced than normally observed with theanticancer drugs used alone in larger doses. Possible dose ranges ofthese co-administered anticancer agents are about 0.1 to 20 mg/kg/day.

The term “co-administration” is meant to refer to any administrationroute in which two or more agents are administered to a patient orsubject. For example, the agents may be administered together, or beforeor after each other. The agents may be administered by different routes,e.g., one agent may be administered intravenously while the second agentis administered intramuscularly, intravenously or orally. The agents maybe administered simultaneously or sequentially, as long as they aregiven in a manner sufficient to allow both agents to achieve effectiveconcentrations in the body. The agents also may be in an admixture, as,for example, in a single tablet. In sequential administration, one agentmay directly follow administration of the other or the agents may begive episodically, i.e., one can be given at one time followed by theother at a later time, typically within a week. An example of a suitableco-administration regimen is where a hypocalcemic vitamin D compound isadministered from 0.5 to 7 days prior to administration of a cytotoxicagent.

Also included within the scope of the present invention is theco-administration of effective dosages of hypercalcemic vitamin Dcompounds in conjunction with administration of hormones or otheragents, e.g., estrogens, which are known to ameliorate bone diseases ordisorders. For example, prostate cancer often metastasizes to bone,causing bone loss and associated pain. Such bone agents may includeconjugated estrogens or their equivalents, calcitonin, bisphosphonates,calcium supplements, cobalamin, pertussis toxin and boron. Possible doseranges for these co-administered bone agents are provided in Table 1.

TABLE 1 Possible Oral Dose Ranges for Various Bone AgentsCo-Administered With 1α-Hydroxyvitamin D of Formula (I) Dose RangesAgent Broad Preferred Most Preferred Conjugated Estrogens or 0.3-5.00.4-2.4 0.6-1.2 Equivalent (mg/day) Sodium Fluoride (mg/day)  5-15030-75 40-60 Calcitonin (IU/day)  5-800  25-500  50-200 Bisphosphonates(mg/day) 0.5-20   1-15  5-10 Calcium Supplements  250-2500  500-1500 750-1000 (mg/day) Cobalamin (μg/day)  5-200  20-100 30-50 PertussisToxin (mg/day)  0.1-2000   10-500  100-1000 Boron (mg/day)  0.10-3000  1-250  2-100 Antiestrogens, such as Tamoxifen ™, are also known boneagents and may be suitably used in conjunction with the1α-hydroxyvitamin D compounds of the present invention.

The present invention is further explained by the following exampleswhich should not be construed by way of limiting the scope of thepresent invention.

VDR BINDING ANALYSES EXAMPLE 1 1α,24-Dihydroxyvitamin D₂ [1α,24-(OH)₂D₂]

The affinity of 1α,24-(OH)₂D₂ for the mammalian vitamin D receptor (VDR)was assessed using a commercially available kit of bovine thymus VDR andstandard 1,25-(OH)₂D₃ solutions from Incstar (Stillwater, Minn.). Thehalf-maximal binding of chemically synthesized 1α,24-(OH)₂D₂ wasapproximately 150 pg/ml whereas that of 1α,25-(OH)₂D₃ was 80 pg/ml.Thus, the 1α,24-(OH)₂D₂ had a very similar affinity for bovine thymusVDR as did 1α,25-(OH)₂D₃, indicating that 1α,24-(OH)₂D₂ has potentbiological activity.

EXAMPLE 2 1α,24-Dihydroxy Vitamin D₄ [1α,24-(OH)₂D₄]

The VDR affinity binding of 1α,24-(OH)₂D₄ was investigated. The1α,24-(OH)₂D₄ was incubated with vitamin D receptor and radiolabeledtracer 1α,25-(OH)₂D₃. After incubation, the amount of radioactivitybound to the receptor was determined and compared with the amount boundafter co-incubation of unlabeled and labeled 1α,25-(OH)₂D₃. It was foundthat 50 pg/tube of 1α,24-(OH)₂D₄ was equivalent to approximately 20 pg1α,25-(OH)₂D₃.

These results show that 1α,24-(OH)₂D₄ binds slightly less tightly to thevitamin D receptor than does 1α,25-(OH)₂D₃. Such data mean that1α,24-(OH)₂D₄ has high affinity for the VDR and significant biologicalactivity, similar to that of 1α,25-(OH)₂D₃. These data are consistentwith gene expression studies done (described below) with 1α,24-(OH)₂D₄which demonstrate that 1α,24-(OH)₂D₄ is only slightly less active thanis 1α,25-(OH)₂D₃.

These results are surprising and unexpected in view of the prior art.They are contrary to the normative wisdom in the vitamin D art regardingthe very low degree of biological activity of vitamin D₄ compounds.

EXAMPLE 3 1α,24-Dihydroxyvitamin D₂ [1α,24-(OH)₂D₂]

VDR binding of vitamin D compounds by prostate cells is demonstratedusing the techniques of Skowronski et al., 136 Endocrinology (1995)20-26, which is incorporated herein by reference. Prostate-derived celllines are cultured to near confluence, washed and harvested by scraping.Cells are washed by centrifugation, and the cell pellet resuspended in abuffered salt solution containing protease inhibitors. The cells aredisrupted by sonication while cooling on ice. The supernatant obtainedfrom centrifuging the disrupted cells at 207,000×g for 35 min at 4EC isassayed for binding. 200 TL of soluble extract, (1-2 mg protein/mlsupernatant) is incubated with a 1 nM ³H-1α,25-(OH)₂D₃ and increasingconcentrations of 1α,24-(OH)₂-D₂ (0.01-100 nM) for 16-20 hr at 4EC.Bound and free hormones are separated with hydroxylapatite usingstandard procedures. Specific binding is calculated by subtractingnonspecific binding obtained in the presence of a 250-fold excess ofnonradioactive 1α,25-(OH)₂D₃ from the total binding measured. Theresults demonstrate that 1α,24-(OH)₂D₂ has strong affinity for prostateVDR, indicating that 1α,24-(OH)₂D₂ has potent biological activity inrespect of prostate cells.

EXAMPLE 4 1α,24-Dihydroxy Vitamin D₄ [1α,24-(OH)₂D₄]

The procedure of Example 3 is repeated using the active vitamin Danalogue 1α,24-(OH)₂D₄, and the specific binding is determined. Theresults demonstrate that 1α,24-(OH)₂D₄ has strong affinity for prostateVDR, indicating that 1α,24-(OH)₂D₄ has potent biological activity inrespect of prostate cells.

EXAMPLE 5 1α,25-Dihydroxyvitamin D₄ [1α,25-(OH)₂D₄]

The procedure of Example 3 is repeated using the active vitamin Danalogue 1α,25-(OH)₂D₄, and the specific binding is determined. Theresults demonstrate that 1α,25-(OH)₂D₄ has strong affinity for prostateVDR, indicating that 1α,25-(OH)₂D₄ has potent biological activity inrespect of prostate cells.

GENE EXPRESSION EXAMPLE 6 1α,24-

Dihydroxy Vitamin D₄ [1α,24-(OH)₂D₄]

Using the plasmids p(CT4)⁴TKGH, a vitamin D receptor (VDR)-expressingplasmid, and pSG5-hVDR1/3, a plasmid containing a Growth Hormone (GH)gene, under the control of a vitamin D-responsive element (VDRE),experiments were conducted to explore the ability of 1α,24-(OH)₂D₄ toinduce vitamin D-dependent growth hormone acting as a reporter genecompared to that of 1α,25-(OH)₂D₃. Cells in culture were transfectedwith these two plasmids. One plasmid contained the gene for GrowthHormone (GH) under the control of the vitamin D responsive element(VDRE) and the other plasmid contained the structural gene for thevitamin D receptor (VDR). These transfected cultures were incubated with1α,24-(OH)₂D₄ or 1α,25-(OH)₂D₃, and the production of growth hormone wasmeasured. Table 2 below shows the results of this assay:

TABLE 2 Induction of Growth Hormone by Vitamin D Compounds ConcentrationGrowth Hormone Compound Used (M) Induction (ng/ml) 1,25-(OH)₂D₃ 1 ×10⁻¹⁰ 39 1,25-(OH)₂D₃ 5 × 10⁻¹⁰ 248 1,24-(OH)₂D₄ 5 × 10⁻¹⁰ 1651,24-(OH)₂D₄ 1 × 10⁻⁹  628 1,24-(OH)₂D₄ 5 × 10⁻⁹  1098

These data show that the ability of 1α,24-(OH)₂D₄ to stimulate vitaminD-dependent growth hormone is nearly equivalent to that of1α,25-(OH)₂D₃. Such results are truly surprising and would not have beenexpected by following the teachings of the prior art.

EXAMPLE 7 1α,24(S)-Dihydroxyvitamin D₂ and 1α,24(R)-Dihydroxy-vitamin D₂[1α,24(S)-(OH)₂D₂ and 1α,24(R)-(OH)₂D₂]

The gene expression study described in Example 6 was conducted tocompare the biological activity in vitro of chemically synthesized1α,24(S)-(OH)₂D₂ and 1α,24(R)-(OH)₂D₂, with 1α,25-(OH)₂D₃ and 25-OH-D₃.The vitamin D-dependent transcriptional activation model system was usedin which plasmids pSG5-hVDR1/3 and p(CT4)⁴TKGH were co-transfected intoGreen monkey kidney, COS-1 cells.

Transfected cells were incubated with vitamin D metabolites and growthhormone production was measured. As shown in Table 3, both1α,24(S)-(OH)₂D₂ and its epimer, 1α,24(R)-(OH)₂D₂, had significantlymore activity in this system than 25-OH-D₃, with 1α,24(S)-(OH)₂D₂ havingnearly the same activity as 1α,25-(OH)₂D₃.

TABLE 3 Vitamin D-Inducible Growth Hormone Production In TransfectedCOS-1 Cells Vitamin DCInducible Growth Hormone Production Net MolarTotal GH vitamin DCinducible Con- Production* GH-production Inducercentration (ng/ml) (ng/ml) Ethanol 44 0 25-OH-D₃ 1 × 10⁻⁷ 245 201 1 ×10⁻⁶ 1100 1056 1 × 10⁻⁵ 775 731 1α,25-(OH)₂D₃ 1 × 10⁻¹⁰ 74 30 1 × 10⁻⁹925 881 1 × 10⁻⁸ 1475 1441 1α,24(S)-(OH)₂D₂ 5 × 10⁻¹⁰ 425 381 5 × 10⁻⁹1350 1306 5 × 10⁻⁸ 1182 1138 1α,24(R)-(OH)₂D₂ 1 × 10⁻⁹ 80 36 1 × 10⁻⁸1100 1056 1 × 10⁻⁷ 1300 1256 *Averages of duplicate determinations

INHIBITION OF CELL PROLIFERATION EXAMPLE 8 1α,24-Dihydroxyvitamin D₂[1α,24-(OH)₂D₂]

Inhibition of cell proliferation is demonstrated using the techniques ofSkowronski et al., 132 Endocrinology (1993) 1952-1960 and 136Endocrinology (1995) 20-26, both of which are incorporated herein byreference. The cell lines, LNCaP and PC-3, which are derived from humanprostate adenocarcinoma, are seeded in six-well tissue culture plates ata density of about 50,000 cells/plate. After the cells have attached andstabilized, about 2-3 days, the medium is replenished with mediumcontaining vehicle or the active vitamin D analogue 1α,24-(OH)₂D₂, atconcentrations from 10⁻¹¹ M to 10⁻⁷ M. Medium containing test analogueor vehicle is replaced every three days. After 6-7 days, the medium isremoved, the cells are rinsed, precipitated with cold 5% trichloroaceticacid, and washed with cold ethanol. The cells are solubilized with 0.2 Nsodium hydroxide, and the amount of DNA determined by standardprocedures. The results show that cultures incubated with 1α,24-(OH)₂D₂in accordance with the present invention have significantly fewer cellsthan the control cultures.

EXAMPLE 9 1α,24-Dihydroxy Vitamin D₄ [1α,24-(OH)₂D₄]

The procedure of Example 8 is repeated using the active vitamin Danalogue 1α,24-(OH)₂D₄, and the cell number is determined. Culturesincubated with 1α,24-(OH)₂D₄ have significantly fewer cells than thecontrol cultures.

EXAMPLE 10 1α,25-Dihydroxyvitamin D₄ [1α,25-(OH)₂D₄]

The procedure of Example 8 is repeated using the active vitamin Danalogue 1α,25-(OH)₂D₄, and the cell number is determined. Culturesincubated with 1α,25-(OH)₂D₄ have significantly fewer cells than thecontrol cultures.

STIMULATION OF CELL DIFFERENTIATION EXAMPLE 11 1α,24-Dihydroxyvitamin D₂[1α,24-(OH)₂D₂]

Using the techniques of Skowronski et al., 132 Endocrinology (1993)1952-1960 and 136 Endocrinology (1995) 20-26, both of which areincorporated herein by reference, cells of the cell line, LNCaP, whichis derived from a human metastatic prostate adenocarcinoma and known toexpress PSA, are seeded in six-well tissue culture plates at a densityof about 50,000 cells/plate. After the cells have attached andstabilized, about 2-3 days, the medium is replenished with mediumcontaining vehicle or the active vitamin D analogue, 1α,24-(OH)₂D₂, atconcentrations from 10⁻¹¹ M to 10⁻⁷ M. After 6-7 days, the medium isremoved and stored at −20EC for prostate specific antigen (PSA)analysis.

The cells from parallel cultures are rinsed, precipitated, and theamount of DNA determined by standard procedures. PSA is measured bystandard known methods. Cultures incubated with 1α,24-(OH)₂D₂ havesignificantly more PSA than control cultures when expressed as mass ofPSA/cell.

EXAMPLE 12 1α,24-Dihydroxyvitamin D₄ [1α,24-(OH)₂D₄]

The procedure of Example 12 is repeated except the active vitamin Danalogue is 1α,24-(OH)₂D₄. The PSA is measured and cultures incubatedwith 1α,24-(OH)₂D₄ have significantly more PSA than control cultureswhen expressed as mass of PSA/cell.

EXAMPLE 13 1α,25-Dihydroxy Vitamin D₄ [1α,24-(OH)₂D₄]

The procedure of Example 12 is repeated except the active vitamin Danalogue is 1α,25-(OH)₂D₄. The PSA is measured and cultures incubatedwith 1α,25-(OH)₂D₄ have significantly more PSA than control cultureswhen expressed as mass of PSA/cell.

CLINICAL STUDIES EXAMPLE 14 General Treatment of Cancers

Patients with a known vitamin D receptor positive tumor (e.g.,adenocarcinoma of the prostate, breast, lung, colon or pancreas, ortransitional cell carcinoma of the bladder, or melanoma) participate inan open-label study of a hypocalcemic vitamin D compound in accordancewith the present invention. Patients are placed on a reduced calciumdiet prior to treatment, to help minimize intestinal absorption andallow ever higher doses of the hypocalcemic vitamin D. This reducedcalcium diet may be continued for the duration of treatment, and for oneweek after the last dose of the 1α,24(S)-dihydroxyvitamin D₂. The dietideally restricts daily calcium intake to 400-500 mg. Patients alsodiscontinue use of any vitamin D supplements or vitamin D replacementtherapies. Each patient is also asked to drink 4-6 cups of fluid morethan usual intake to assure adequate oral hydration.

Each subject is monitored at regular intervals for: (1) hypercalcemia,hyperphosphatemia, hypercalciuria, hyperphosphaturia and other toxicity;(2) evidence of changes in the progression of metastatic disease; and(3) compliance with the prescribed test drug dosage.

The dosing regimen is typically on a daily dose basis of 10 μg or 20 μgper day to about 100 μg/day for 24 months. Alternatively, a non-dailydosing regimen can be used, e.g., 40 μg given every other day, 100 μggiven once a week. The route of administration can vary from oral tointravenous to regional delivery (e.g., arterial infusion, via theportal vein). Oral is, of course, the easiest and most cost effectiveroute. Regional delivery permits high dosing and generally avoids anyproduction of hypercalcemia. Although, in the case of the compound ofthe present invention, the compound is substantially hypocalcemic.

After 18 months of treatment, CAT, scans, X-rays and bone scans used forevaluating the progress of metastatic disease or partial remission inmany patients treated at the lower dosage, and stable disease andpartial or complete remission in many patients treated at the higherdosage.

EXAMPLE 15 Treatment of Prostate Cancer with 1α,24-Dihydroxy Vitamin D₂[1α,24-(OH)₂D₂]

Patients with advanced androgen-independent prostate cancer participatein an open-labeled study of 1α,24-(OH)₂D₂. Qualified patients are atleast 40 years old, exhibit histologic evidence of adenocarcinoma of theprostate, and present with progressive disease which had previouslyresponded to hormonal intervention(s). On admission to the study,patients begin a course of therapy with oral 1α,24-(OH)₂D₂ lasting 26weeks, while discontinuing any previous use of calcium supplements,vitamin D supplements, and vitamin D hormone replacement therapies.During treatment, the patients are monitored at regular intervals for:(1) hypercalcemia, hyperphosphatemia, hypercalciuria, hyperphosphaturiaand other toxicity; (2) evidence of changes in the progression ofmetastatic disease; and (3) compliance with the prescribed test drugdosage.

The study is conducted in two phases. During the first phase, themaximal tolerated dosage (MTD) of daily oral 1α,24-(OH)₂D₂ is determinedby administering progressively higher dosages to successive groups ofpatients. All doses are administered in the morning before breakfast.The first group of patients is treated with 25.0 μg of 1α,24-(OH)₂D₂.Subsequent groups of patients are treated with 50.0, 75.0 and 100.0μg/day. Dosing is continued uninterrupted for the duration of the studyunless serum calcium exceeds 11.6 mg/dL, or other toxicity of grade 3 or4 is observed, in which case dosing is held in abeyance until resolutionof the observed toxic effect(s) and then resumed at a level which hasbeen decreased by 10.0 μg.

Results from the first phase of the study show that the MTD for1α,24-(OH)₂D₂ is above 20.0 μg/day, a level which is 10- to 40-foldhigher than can be achieved with 1α,25-(OH)₂D₃. Analysis of bloodsamples collected at regular intervals from the participating patientsreveal that the levels of circulating 1α,24-(OH)₂D₂ increaseproportionately with the dosage administered, rising to maximum levelswell above 100 pg/mL at the highest dosages, and that circulating levelsof 1α,25-(OH)₂D₃ are suppressed, often to undetectable levels. Serum andurine calcium are elevated in a dose responsive manner. Patients treatedwith the MTD of 1α,24-(OH)₂D₂ for at least sixmonths report that bonepain associated with metastatic disease is significantly diminished.

During the second phase, patients are treated with 1α,24-(OH)₂D₂ for 24months at 0.5 and 1.0 times the MTD. After one and two years oftreatment, CAT scans, X-rays and bone scans used for evaluating theprogression of metastatic disease show stable disease or partialremission in many patients treated at the lower dosage, and stabledisease and partial or complete remission in many patients treated atthe higher dosage.

EXAMPLE 16 Treatment of Prostate Cancer with 1α-Hydroxy Vitamin D₂[1α-OH-D₂]

The study of Example 14 is repeated for the active vitamin D compound,1α-OH-D₂. The results of the phase one study indicate that patientstreated with the MTD of 1α-OH-D₂ for at least sixmonths report that bonepain associated with metastatic disease is significantly diminished. Theresults of the phase two study indicate that after two years, CAT scans,X-rays and bone scans used for evaluating the progression of metastaticdisease show stable disease or partial remission in many patientstreated at the lower dosage, and stable disease and partial or completeremission in many patients treated at the higher dosage.

EXAMPLE 17 Treatment of Melanoma

The method of Example 14 is used to treat patients with metastaticmalignant melanoma of, e.g., the jaw. After 18 months of treatment, theprogress of the metastatic disease shows stable disease or partialremission.

EXAMPLE 18 Treatment of Retinoblastoma

The method of Example 14 is used is used to treat patients withmetastatic retinoblastoma. After 18 months of treatment, the progress ofthe metastatic disease shows stable disease or partial remission.

EXAMPLE 19 Treatment of Liver Cancer

The method of Example 14 is used to treat patients with hepatoma. Theregional delivery of the compound in accordance with the presentinvention, i.e., via arterial infusion, is used. After 18 months oftreatment, the progress of the metastatic disease shows stable diseaseor partial remission.

While the present invention has now been described and exemplified withsome specificity, those skilled in the art will appreciate the variousmodifications, including variations, additions, and omissions, that maybe made in what has been described. Accordingly, it is intended thatthese modifications also be encompassed by the present invention andthat the scope of the present invention be limited solely by thebroadest interpretation lawfully accorded the appended claims.

What is claimed is:
 1. A method of inhibiting hyperproliferation ofmalignant or neoplastic cells, comprising treating the cells with anantiproliferative amount of a hypocalcemic hydroxyvitamin D compoundhaving a hydrocarbon moiety at the C₂₄ position, the cells expressing avitamin D receptor.
 2. A method in accordance with claim 1, wherein thecells are cancers of the breast, colon, lung, neck and head, pancreas,endometrium, bladder, cervix, testes, ovaries, and liver, squamous cellcarcinoma, myeloid and lymphocytic leukemia, lymphoma, medullary thyroidcarcinoma, melanoma, multiple myeloma, retinoblastoma or sarcomas of thesoft tissues and bone.
 3. A method in accordance with claim 1, whereinthe hypocalcemic vitamin D is a compound represented by formula (I)

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alky, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl group, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes abond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or,taken with R¹ or R², constitutes a double bond, and X³ is hydrogen orhydroxyl provided that at least one of X¹, X² and X³ is hydroxyl; and Yis a methylene group if the bond to Y is a double bond or is a methylgroup or hydrogen if the bond to Y is a single bond.
 4. A method inaccordance with claim 1 wherein the hypocalcemic vitamin D compound is acompound of formula II

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl group, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes abond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or,taken with R¹ or R², constitutes a double bond, and Y is a methylenegroup if the bond to Y is a double bond or is a methyl group or hydrogenif the bond to Y is a single bond.
 5. A method in accordance with claim1, wherein the hypocalcemic vitamin D compound is a compound of formulaIII:

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl group, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes abond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or,taken with R¹ or R², constitutes a double bond.
 6. A method ofinhibiting the hyperproliferative activity of malignant or neoplasticcells, comprising administering to a patient suffering therefrom, ananitproliferarive amount of a hypocalcemic hydroxyvitamin D compound. 7.A method in accordance with claim 6, wherein the hypocalcemic vitamin Dcompound is administered in a daily regimen or an episodic regimen.
 8. Amethod in accordance with claim 7, wherein the episodic regimen is adose once every 2 to 7 days.
 9. A method in accordance with claim 7,wherein the hypocalcemic vitamin D compound is administered daily at adose of about 10 to 100 μg/day.
 10. A method in accordance with claim 6,wherein the hypocalcemic vitamin D compound is administered orally, isadministered intravenously, is directly injected to a cancer site or isregionally delivered to a cancer site.
 11. A method in accordance withclaim 10, wherein the hypocalcemic vitamin D compound is administeredorally.
 12. A method in accordance with claim 6, wherein thehypocalcemic vitamin D compound is co-administered with a cytotoxicagent.
 13. A method in accordance with claim 12, wherein the cytotoxicagent is an antimetabolite, and antimicrotubule agent, an alkyatingagent, a platinum agent, an anthracycline, a topoisomase inhibitor, oran antibiotic.
 14. A method in accordance with claim 13, wherein theantimetabolite is 5-fluoro-uracil, methotrexate or fludarabine.
 15. Amethod in accordance with claim 13, wherein the antimicrotubule agent isvincristine, vinblastine or a taxane.
 16. A method in accordance withclaim 14, wherein the taxane is paclitaxel or docetaxel.
 17. A method inaccordance with claim 12, wherein the alkylating agent iscyclophasphamide, melphalan, biochoroethylnitrosurea or hydroxyurea. 18.A method in accordance with claim 12, wherein the platinum agent iscisplatin, carboplatin, oxaliplatin, JM-216 or CI-973.
 19. A method inaccordance with claim 12, wherein the anthracycline is doxrubicin ordaunorubicin.
 20. A method in accordance with claim 12, wherein theantibiotic is mitomycin, idarubicin, adriamycin or daunomycin.
 21. Amethod in accordance with claim 12, wherein the topoisomerase inhibitioris etoposide or camptothecins.
 22. A method in accordance with claim 12,wherein the cytotoxic agent is estramustine phosphate or prednimustine.23. A method in accordance with claim 11, wherein an antiproliferativeeffective amount of the cytotoxic agent is lower than theantiproliferative effective amount of the cytotoxic agent whenadministered alone.
 24. The method of claim 5, wherein the compound offormula (III) is 1α,24-dihydroxyvitamin D₂, 1α,24-dihydroxyvitamin D₄,1α,25-dihydroxyvitamin D₂, 1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitaminD₂ or 1α-hydroxyvitamin D₄.
 25. A method of treating a human toalleviate the pathological effects of breast cancer, colon cancer,testicular cancer, pancreatic cancer, endometrial cancer, small cell andnon-small cell cancer of the lung (including squamous, adneocarcinomaand large cell types), squamous cell of the head and neck, bladder,ovarian and cervical cancers, myeloid and lymphocyltic leukemia,lymphoma, hepatic tumors, medullary thyroid carcinoma, multiple myeloma,melanoma, retinoblastoma or sarcomas of the soft tissue and bone,comprising administering to the human an therapeutic amount of ahypocalcemic hydroxyvitamin D compound.
 26. A method of claim 25,wherein said hypocalcemic vitamin D is a 1α-hydroxyvitamin D compoundrepresented by formula (III)

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl group, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes abond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or,taken with R¹ or R², costitutes a double bond.
 27. The method of claim26, wherein said therapeutic amount is 0.01 μg/kg/day to 2.0 μg/kg/day.28. The method of claim 26, wherein the compound of formula (III) is1α,24-dihydroxyvitamin D₂, 1α,24-dihydroxyvitamin D₄,1α,25-dihydroxyvitamin D₂, 1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitaminD₂ or 1α-hydroxyvitamin D₄.
 29. A method of enhancing theantiproliferative effect of a cytotoxic agent in a patient with adisease in need of treatment by a cytotoxic agent, comprisingadministering to the patient a therapeutic amount of hypocalcemicvitamin D compound and the cytotoxic agent.
 30. A method in accordancewith claim 29, wherein the hypocalcemic vitamin D compound isadministered from 0.5 to 7 days prior to administration of the cytotoxicagent.
 31. A method in accordance with claim 29, wherein thehypocalcemic vitamin D compound is administered 2 to 4 days prior toadministration of the cytotoxic agent.
 32. A method of claim 29, whereinsaid hypocalcemic vitamin D is a 1α-hydroxyvitamin D compoundrepresented by formula (III)

wherein A¹ and A² each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl group, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes abond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or,taken with R¹ or R², constitutes a double bond.
 33. The method of claim32, wherein said therapeutic amount of the vitamin D compound is 0.01μg/kg/day to 2.0 μg/kg/day.
 34. The method of claim 32, wherein thecompound of formula (III) is 1α,24-dihydroxyvitamin D₂,1α,24-dihydroxyvitamin D₄, 1α25-dihydroxyvitamin D₂,1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitamin D₂ or 1α-hydroxyvitamin D₄.35. A method in accordance with claim 32, wherein the cytotoxic agent isan antimetabolite, and antimicrotubule agent, an alkyating agent, aplatinum agent, an anthracycline, a topoisomase inhibitor, or anantibiotic.
 36. A method of inducing differentiation in malignant orneoplastic cells, comprising treating to the cells with aprodifferentiative amount of a hypocalcemic vitamin D compound.
 37. Amethod of treating in a subject tumor or neoplasm that expresses avitamin D receptor, comprising administering to the subject an effectiveamount of hypocalcemic vitamin D compound sufficient to raise a bloodlevel of vitamin D to a sufficiently supraphysiological level for asufficient period of time to inhibit growth of the tumor or neoplasmwithout causing hypercalcemia in the subject.
 38. A method in accordancewith claim 7, wherein the hypocalcemic vitamin D compound isadministered episodically at a dose of about 10 μg to 200 μg/dose.
 39. Amethod in accordance with claim 8, wherein the hypocalcemic vitamin Dcompound is administered at a dose of about 10 μg to 200 μg/dose.
 40. Amethod in accordance with claim 25, wherein the pathological effects aredue to hepatic tumors.
 41. A method in accordance with claim 25, whereinthe pathological effects are due to retinoblastoma.
 42. A method inaccordance with claim 25, wherein the therapeutic amount is administeredepisodically.
 43. A method in accordance with claim 42, wherein thetherapeutic amount is 10 μg to 200 μg/dose.
 44. A method in accordancewith claim 29, wherein the therapeutic amount of the hypocalcemicvitamin D compound is 10 μg to 200 μg/dose.